A stepwise method development strategy was employed in developing a robust HPLC method to resolve several closely eluting process impurities associated with a novel diabetes compound. The strategy consisted of rapid column screening, optimization of mobile phase compositions and separation temperature, DryLab modelling, and experimental verification of optimized separation conditions.

The column evaluation process involved screening of a series of 20 columns varying in bonding chemistry using four sets of mobile phases composed of water, acetonitrile and/or methanol at three different pHs. The screening process resulted in identifying two promising columns: XBridge Shield RP18 and SunFire C18. The effects of organic modifiers and separation temperatures were then evaluated to narrow down the chromatographic separation parameters. DryLab� was used to predict optimized gradient profile and separation temperature. Finally, the DryLab� predictions were verified experimentally.

The study demonstrates that factors such as stationary phase composition, organic modifiers, pH and separation temperature have profound and oftentimes complex effects on chromatographic conditions. Therefore, it is critical to adopt a rational strategy as demonstrated here to evaluate the interplay of these factors, there by greatly enhancing method development efficiency.